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Plants can produce diverse groups of secondary metabolites to adapt to environment. Many secondary metabolites are involved in the defense responses against pathogenic microbes, including phytoanticipins which are low molecular weight anti-microbial compounds presented in plants before infection, and phytoalexins produced by plants de novo in response to pathogen attack. Phytoalexins are an important part of plant defense repertoire to pathogenic microbes, especially to necrotrophs. Since the concept of phytoalexin was proposed 80 years ago, many kinds of phytoalexins were identified. In contrast, the biosynthesis of most phytoalexins and their regulatory networks are largely unknown. In this review, I summarized recent research progress of phytoalexins in Arabidopsis and Nicotiana species, with special focus on molecular regulations of their biosynthesis. The problems and future directions in phytoalexin research were also discussed.